We are using 3T3-L1 fat cells for studying hormonal regulation of lipoprotein lipase (LPL) in cells. We recently developed a radioimmunoassay for the enzyme, which allows comparison of the amount of lipolytic activity with the amount of immunoreactive protein (IRP) present in cells and in the medium under various conmditions. Our findings indicate IRP probably exists in two forms in the cells: one that is releasable by heparin and had lipolytic activity, and another that is not releasable by heparin, has little or no lipolytic activity, is probably membrane associated, and is assayed as IRP only after being solubilized by SDS. We earlier reported that LPL activity in the medium is increased when cells are treated with insulin. Our recent findings show this increase in LPL activity is paralleled by a comparable increase in IRP. Studies with radioactive methionine showed that insulin stimulates synthesis of lipoprotein lipase. Cyclohexamide prevented increased in LPL activity and IRP, but did not affect the amount of LPL activity released from cells by heparin. Incubation with insulin for 1 hr increased 5-fold the amount of LPL activity released from cells by heparin, suggesting that insulin is also involved in transport of the enzyme within fat cells.